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Am. J. Pathol.171, 1153-1167.
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.2007

Notes: To study how chemotherapy affects hair follicles (HF), the researchers microdissected and cultured human anagen VI scalp follicles and treated the cells with 4-Hydroperoxycyclophosphamide (4-HC) at similar concentrations to those received during therapy. After incubation for 24 hours with 30µmol/L of 4-HC, the HFs were homogenized and genomic DNA was purified using the Wizard® SV Genomic DNA Purification System. To determine if the treatment induced the mitochondrial common DNA deletion, the isolated DNA was subjected to real-time PCR. Further examination of gene expression was performed by isolating total RNA from two HF samples, reverse transcribing 3µg of the RNA using 15U of AMV Reverse Transcriptase and 0.025 µg/µl random primers, and amplifying seven human genes in a TaqMan® Assay. (3746)

Notes: The authors characterized expression of extracellular proteins by white-rot fungus, Phanerochaete chrysosporium, grown on wood. Temporal expression of these proteins was monitored by relative quantitative RT-PCR. Two micrograms of total RNA was reversed transcribed using 1µg of Random Primers at 37°C for 1 hour. PCRs with one set of PCR primers were performed using 0.5 units of GoTaq® DNA Polymerase, 1X reaction buffer, 250µM each dNTP, 0.5µM each primer and 1µl of cDNA. PCRs with two sets of PCR primers were performed using 2.5 units of GoTaq® DNA Polymerase, 1.6X reaction buffer, 500µM each dNTP, 0.5µM each primer and 1µl of cDNA. (3708)

Notes: The authors investigated the role of estrogen inactivation by the SULT1E1-encoded estrogen sulfotransferase in ovulation in mice. Semiquantitative RT-PCR was used to characterize the temporal and tissue-specific expression of SULT1E1 mRNA in ovulating mice. First-strand cDNA synthesis was performed at 37°C for 2 hours using 7.5µg of total RNA, 1µl (0.5µg) of oligo(dT)15, 40 units of RNasin® Ribonuclease Inhibitor and 200 units of M-MLV Reverse Transcriptase. (3913)

Notes: The authors investigated the effect of neonatal bisphenol A (BPA) exposure in rats on expression of estrogen receptor α (ERα) transcripts. Alternative ERα transcripts in preoptic area of treated and untreated rats were quantified using real-time RT-PCR. Reverse transcription was performed using 4µg of total RNA, 200pmol random primers and 300 units M-MLV Reverse Transcriptase. Real-time PCR was performed using SYBR® Green I to quantify amplified products. To determine if the changes in BPA-induced ERα transcript expression were caused by DNA methylation, the methylation status of the five ERα promoters was examined by bisulfite modification. Genomic DNA was isolated from rat tissue using the Wizard® Genomic DNA Purification Kit, denatured with NaOH, then treated with hydroquinone and sodium bisulfite. Prior to methylation-specific PCR, DNA was cleaned up using the Wizard® DNA Purification Resin as directed by the manufacturer. PCR products were cleaned up again using the Wizard® SV Gel and PCR Clean-Up System, then subjected to restriction enzyme digestion and agarose gel electrophoresis to reveal methylation-dependent sequence differences. (3911)

Notes: Two genes (CzcR and CzcS) encoding the CzcCBA (cobalt/zinc/cadmium) heavy metal efflux pump were cloned using Pfu DNA Polymerase and genomic DNA from PT5 and PT1105 strains of Pseudomonas aeruginosa. The products were 900 and 1,600 bp, respectively. RNA from 5 different strains of Pseudomonas aeruginosa was isolated and treated with RQ1 RNase-Free DNase. The RNA was used in reverse transcription reactions with the ImProm-II™ Reverse Transcriptase and random primers. Copy-DNAs produced from the reaction were stored at -20°C until use in real-time PCR. (3044)

Notes: These authors performed gel shift (EMSA) assays to determine whether purified VirA binds to the vapA promoter. Radiolabeled DNA fragments for the EMSA assays were prepared using DNA Polymerase I Large (Klenow) Fragment. Primer extension using ImProm-II™ Reverse Transcriptase localized the transcription start site within the vapA promoter. To characterize the transcriptional organization of the virR gene cluster, the authors performed reverse transcription using ImProm-II™ Reverse Transcriptase and Random Primers, followed by PCR using a combinations of primers in opposite orientations throughout the gene cluster. The plasmids used in this study were purified by alkaline lysis method or using the Wizard®Plus SV Minipreps DNA Purification System. (3563)

Notes: RNA was extracted from 300mg malignant mesothelioma tissue samples and treated with DNase in the presence of RNasin® Ribonuclease Inhibitor. The RNA was incubated with oligo(dT) primers and reverse transcribed in the presence of radiolabeled nucleotides. The labeled cDNA was hybridized to preprinted microarrays containing 4132 human genes. (2715)

Notes: Total and poly(A)+ RNA from treated and untreated 8701-BC breast cancer tumor cells were treated with RQ1 RNase-free DNase. The RNA samples were reverse transcribed using the M-MLV RNase H– point mutant reverse transcriptase and random primers to create cDNA used in downstream analyses. The cDNA from the reactions were used in PCR, differential display PCR, and semi-quantitative multiplex PCR reactions. In differential display PCR studies, the authors analyzed differentially displayed bands by staining 6% polyacrylamide gels with the SILVER SEQUENCE™ Staining Reagents. Differentially displayed bands were re-amplified and cloned using the pGEM®-T Easy Vector and high efficiency JM109 competent cells. (3020)

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